Isolation of fibroblast growth factor

Abstract

Basic Fibroblast Growth Factor (FGF) is substantially purified by the employment of affinity chromatography using heparin-linked support material. Described is a simplified three step procedure for extracting basic FGF from either mammalian brain or mammalian pituitary tissue. Salt precipitation, e.g., with ammonium sulfate is used to provide a partially purified precipitate that is then subjected to ion-exchange chromatography, e.g., using a Carboxymethyl-Sephadex column. Substantially pure basic FGF fractions are then obtained by fractionating the further partially purified fractions using affinity chromatography on a heparin-linked support e.g., Heparin-Sepharose.

What is claimed is: 
     
       1. A method for separating basic fibroblast growth factor (bFGF) from a biological sample, said method comprising the steps of: binding bFGF in the biological sample to heparin linked to an insoluble support; eluting bFGF from said linked heparin by gradient fractionation; and selecting for fractions capable of inducing proliferation of vascular endothelial cells in vitro. 
     
     
       2. A method according to claim 1 wherein the eluted bFGF exhibits an increased purity of at least about 2400 to 3500 fold, compared to the biological sample. 
     
     
       3. A method according to claim 1, wherein the eluted bFGF comprises at least about 90 percent by weight of protein in the eluent from the gradient fractionation. 
     
     
       4. A method according to claim 1, wherein the biological sample is pre-purified to contain at least about 0.02 to 0.05 percent bFGF by weight of total protein. 
     
     
       5. A method according to claim 4, wherein the pre-purification includes a salt precipitation step. 
     
     
       6. A method according to claim 5, wherein the salt is ammonium sulfate, at a concentration of about 0.15M. 
     
     
       7. A method according to claim 5, wherein the pe-purification includes a step for removal of neutral or acidic proteins in the biological sample. 
     
     
       8. A method according to claim 7, wherein the removal step utilizes an ion exchange resin. 
     
     
       9. A method according to claim 4, wherein the biological sample is an organ tissue selected from the group consisting essentially of brain, pituitary gland, adrenal gland, kidney and placenta. 
     
     
       10. A method according to claim 1, wherein the gradient fractionation utilizes a NaCl salt gradient of 1.1 M to 2 M.